| Part ?:Extracellular vesicles derived from LLC cells inhibit the m TOR pathway and induce C2C12 myotube atrophyBackground Cancer cachexia is a cancer-associated complex metabolic disorder characterized by sustained skeletal muscle depletion,which could not be completely relieved by conventional nutrition support.Extracellular vesicles(EVs)are vesicle-like bodies secreted by cells or detached from the cell membrane.This study was to investigate the biological effects of EVs derived from LLC cells on C2C12 myotubes.Methods C2C12 cells were induced into C2C12 myotubes by differentiation medium(DMEM medium containing 2%horse serum).Lewis lung cancer cells(LLC)and lung epithelial cells(MLE-12)were cultured in the serum-free medium for 48 hours to obtain LLC supernatant and MLE-12 supernatant.The MLE-12 or LLC conditioned medium(CM)was obtained by mixing the MLE-12 or LLC supernatant with DMEM medium at a ratio of1:3,followed by adding 2%horse serum.Extra PEG method was used to extract EVs in the supernatant.EVs were then identified by transmission electron microscopy,particle size measurement,and Western Blot.PKH26 was used to trace the EVs.Immunofluorescence of myosin heavy chain protein(My HC)was used to show the morphology of C2C12myotubes.Western Blot was used to detect the expression levels of My HC,rapamycin target protein(m TOR),phosphorylated m TOR(p-m TOR),F-box protein 32(Fbx32),p70ribosomal protein S6 kinase(p70S6K),and phosphorylated p70S6K(p-p70S6K)in myotubes.Results There were no significant changes in the average diameter of C2C12 myotubes,the expression levels of My HC,m TOR,p-m TOR,Fbx32 and the relative expression of p-p70S6K between the normal C2C12 myotubes and C2C12 myotubes treated by MLE-12CM.Compared with the above two groups,C2C12 myotubes treated by LLC CM had significantly decreased expression levels of My HC,m TOR,p-m TOR,relative p-p70S6K and increased Fbx32 protein expression.Depleting EVs in LLC supernatant could significantly alleviate myotube atrophy induced by LLC CM.The LLC-derived EVs were identified to conform to the features of exosomes.The PKH26-labelled EVs were taken by C2C12 myotubes in vitro.LLC-derived EVs could directly reduce the average diameter of C2C12 myotubes,decrease the expression levels of My HC,m TOR,p-m TOR and relative p-p70S6K,and promote Fbx32 expression in C2C12 myotubes.Conclusion Extracellular vesicles secreted by LLC cells could down-regulate the m TOR pathway in C2C12 myotubes and induce myotube atrophy.Part ?:Identification of the mi R-199a-3p in extracellular vesicles as a biomarker of lung cancer cachexiaBackground The clinical diagnosis of cachexia(more than 5%weight loss in the last 6months or more than 2%weight loss in individuals with sarcopenia/BMI<20 kg/m~2 in the last 6 months)ignore the early pathological changes of cachexia.Exploring potential biomarkers of cancer cachexia for early diagnosis is still urgent.As a kind of"liquid biopsy",extracellular vesicles(EVs)detection has not been proved practicable in the diagnosis of cancer cachexia.This study was to explore appliciable mi RNA in plasma EVs served as a potential biomarker of cancer cachexia.Methods The mi RNA sequencing data of cachectic muscle tissue(GSE75473)and LLC-derived EVs(GSE80678)in GEO database were downloaded and analyzed to explore mi RNAs that potentially participate in muscle atrophy.RT-q PCR was used to investigate the expression levels of mi RNAs in EVs and C2C12 myotubes.The relationship between mi RNA expression and the prognosis of lung cancer patients was investigated using the GEO database and TCGA database.For the cross-sectional study,a total of 51 primary lung cancer patients without any anti-cancer treatments from Tongji Hospital of Huazhong University of Science and Technology and The First Affiliated Hospital of Anhui Medical University from June 2019 to November 2019 were included.The general characteristics,clinical information,and plasma were collected.EVs in plasma were extracted using commercial kits.The clinically applicable cachexia staging(CCS)score was used to assess the severity of cachexia in lung cancer patients.Results Bioinformatics analysis showed that mi R-199a-3p expression in cachectic muscle was 1.498 times that of non-cachectic muscle.Mir-199a,the precursor of mi R-199a-3p,was existed in LLC-derived EVs with high abundance.RT-q PCR then validated the existence of mi R-199a-3p in LLC-derived EVs.The LLC conditioned medium significantly increased the expression level of mi R-199a-3p in the C2C12 myotubes.Though analyzing GEO and TCGA databases,mi R-199a-3p expression was found to be significantly higher in lung cancer tissues than that in normal tissues.However,mi R-199a-3p expression in lung cancer tissues did not have prognostic significance.The expression levels of mi R-199a-3p in EVs(EVs-mi R-199a-3p)in plasma were not correlated with gender,age,smoking,pathological type,tumor stage,and body mass index,but was significantly correlated with ECOG score,anorexia score,and cachexia in lung cancer patients.The EVs-mi R-199a-3p expression of lung cancer patients with cachexia was significantly higher than that of patients without cachexia.The area under the ROC curve of EVs-mi R-199a-3p in diagnosing lung cancer cachexia was 0.710(95%CI:0.5692-0.851).EVs-mi R-199a-3p expression of lung cancer patients with CCS score greater than or equal to 5 points was significantly higher than that of lung cancer patients with CCS score less than 5 points.Besides,expression levels of EVs-mi R-199a-3p in plasma were positively correlated to the CCS scores with statistical significance.Conclusion Mi R-199a-3p in plasma extracellular vesicles is associated with lung cancer cachexia and the severity of cachexia.Part ?:The mi R-199a-3p in extracellular vesicles derived from LLC cells inhibits m TOR pathway and induces cachectic muscle atrophyBackground Extracellular vesicles(EVs)provide stable conformational space and biological protection for inside proteins and nucleic acids,thus participate in a series of tumor-related pathophysiological processes through transferring biological information and materials.When the m TOR signaling in muscle is inhibited,the protein synthesis and multiple myogenic determinant proteins are suppressed,leading to the imbalance of anabolism and catabolism.Mi R-199a-3p was bioinformatically predicted to combind with the 3'UTR region of m TOR m RNA.This study aimed to investigate whether mi R-199a-3p in LLC EVs participate in the muscle wasting in cancer cachexia.Methods The interaction between mi R-199a-3p and m TOR m RNA was investigated by the double luciferase reporter gene assay.The mi R-199a-3p mimics were transfected into C2C12 cells to increase the level of mi R-199a-3p in myotubes.The FAM and PKH26 were used to trace mi R-199a-3p and extracellular vesicles respectively.Lentivirus vectors were used to construct the stably mi R-199a-3p-overexpressed LLC cells(LLC-mi R)and the negative control cells(LLC-NC).Western Blot was used to detect the change of protein levels in C2C12 myotubes treated by LLC-mi R-derived EVs(EVs-mi R)and LLC-NC-derived EVs(EVs-NC).For in vivo study,C57BL/6(6-week-old)mice were randomly divided into four groups:The Ctrl group without any treatment;The LLC group with subcutaneous LLC inoculation followed by normal saline i.p.daily;The LLC-mi R group with subcutaneous LLC-mi R inoculation followed by normal saline i.p.daily;The LLC-mi R+Salidr group with subcutaneous LLC-mi R inoculation followed by 100mg/kg salidroside i.p.daily.Plasma was collected through the retro-orbital sinus 25 days after tumor inoculation.The subcutaneous tumor and bilateral gastrocnemius muscles were integrally harvested and weighed.HE staining was used to evaluate the cross-sectional area of gastrocnemius muscle fibers.The expression changes of the protein and m RNA in gastrocnemius muscle were detected by Western Blot and RT-q PCR.Results The mi R-199a-3p could combind with the 124-130 sites of the m TOR 3'UTR region.Overexpression of mi R-199a-3p significantly reduced the average diameter of C2C12 myotubes and inhibited the protein expression levels of My HC,m TOR,p-m TOR.FAM-labeled mi R-199a-3p in PKH26-labelled EVs could be absorbed into C2C12myotubes.Compared with the LLC-NC supernatant,LLC-mi R supernatant further inhibited the average diameter of C2C12 myotubes,My HC expression and m TOR signaling pathway,indicating LLC-mi R cells possessed stronger pro-cachectic effect.GW4869 treatment significantly reduced the EVs content and pro-cachectic effects of LLC-mi R supernatant.Compared with EVs-NC treatment,the EVs-mi R treatment significantly elevated the expression level of mi R-199a-3p in C2C12 myotubes,inhibited My HC expression and the m TOR signaling pathway.Activation of the m TOR pathway by salidroside significantly alleviated the EVs-NC-inducing and EVs-mi R-aggravating atrophy of C2C12 myotubes.Compared with the Ctrl group,the tumor-free body weight,gastrocnemius mass,and gastrocnemius cross-sectional area were decreased,the m TOR signaling was inhibited,mi R-199a-3p levels in plasma EVs and muscle tissues were elevated in tumor-bearing mice of the LLC group.Compared with the LLC group,the expression levels of mi R-199a-3p in plasma EVs and muscle tissue were significantly increased in the LLC-mi R group,accompanied by a further reduction in tumor-free body weight,gastrocnemius mass,gastrocnemius cross-sectional area and a further suppression of m TOR signaling pathway.Salidroside treatment could alleviate the m TOR inhibition in muscle tissues,meanwhile improve the tumor-free body weight,gastrocnemius mass,and gastrocnemius cross-sectional area of LLC-mi R bearing mice.Conclusion Mi R-199a-3p in LLC-derived extracellular vesicles induces cachectic muscle atrophy by inhibiting the m TOR signaling pathway. |
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